The present invention is generally in the field of transgenic plant systems for the production of polyhydroxyalkanoate materials, modification of triglycerides and fatty acids, and methods for altering seed production in plants.
Methods for producing stable transgenic plants for agronomic crops have been developed over the last 15 years. Crops have been genetically modified for improvements in both input and output traits. In the former traits, tolerance to specific agrochemicals has been engineered into crops, and specific natural pesticides, such as the Bacillus thuringenesis toxin, have been expressed directly in the plant. There also has been significant progress in developing male sterility systems for the production of hybrid plants. With respect to output traits, crops are being modified to increase the value of the product, generally the seed, grain, or fiber of the plant. Critical metabolic targets include the modification of starch, fatty acid, and oil biosynthetic pathways.
There is considerable commercial interest in producing microbial polyhydroxyalkanoate (PHA) biopolymers in plant crops. See, for example, U.S. Pat. Nos. 5,245,023 and 5,250,430 to Peoples and Sinskey; U.S. Pat. No. 5,502,273 to Bright et al.; U.S. Pat. No. 5,534,432 to Peoples and Sinskey; U.S. Pat. No. 5,602,321 to John; U.S. Pat. No. 5,610,041 to Somerville et al.; PCT WO 91/00917; PCT WO 92/19747; PCT WO 93/02187; PCT WO 93/02194; PCT WO 94/12014; Poirier et al., Science 256:520-23 (1992); van der Leij and Witholt, Can. J. Microbiol. 41(supplement):222-38 (1995); Nawrath and Poirier, The International Symposium on Bacterial Polyhydroxyalkanoates, (Eggink et al., eds.) Davos Switzerland (Aug. 18-23, 1996); Williams and Peoples, CHEMTECH 26: 38-44 (1996), and the recent excellent review by Madison, L. and G. Husiman, Microbiol. Mol. Biol. 21-53 (March 1999). PHAs are natural, thermoplastic polyesters and can be processed by traditional polymer techniques for use in an enormous variety of applications, including consumer packaging, disposable diaper linings and garbage bags, food and medical products.
Early studies on the production of polyhydroxybutyrate in the chloroplasts of the experimental plant system Arabidopsis thaliana resulted in the accumulation of up to 14% of the leaf dry weight as PHB (Nawrath et al., 1993). Arabidopsis, however, has no agronomic value. Moreover, in order to economically produce PHAs in agronomic crops, it is desirable to produce the PHAs in the seeds, so that the current infrastructure for harvesting and processing seeds can be utilized. The options for recovery of the PHAs from plant seeds (PCT WO 97/15681) and the end use applications (Williams and Peoples, CHEMTECH 26:38-44 (1996)) are significantly affected by the polymer composition. Therefore, it would be advantageous to develop transgenic plant systems that produce PHA polymers having a well-defined composition, as well as produce PHA polymer in specific locations within the plants and/or seeds.
Careful selection of the PHA biosynthetic enzymes on the basis of their substrate specificity allows for the production of PHA polymers of defined composition in transgenic systems (U.S. Pat. Nos. 5,229,279; 5,245,023; 5,250,430; 5,480,794; 5,512,669; 5,534,432; 5,661,026; and 5,663,063).
In bacteria, each PHA group is produced by a specific pathway. In the case of the short pendant group PHAs, three enzymes are involved: xcex2-ketothiolase, acetoacetyl-CoA reductase, and PHA synthase. The homopolymer PHB, for example, is produced by the condensation of two molecules of acetyl-coenzyme A to give acetoacetyl-coenzyme A. The latter then is reduced to the chiral intermediate R-3-hydroxybutyryl-coenzyme A by the reductase, and subsequently polymerized by the PHA synthase enzyme. The PHA synthase notably has a relatively wide substrate specificity which allows it to polymerize C3-C5 hydroxy acid monomers including both 4-hydroxy and 5-hydroxy acid units. This biosynthetic pathway is found in a number of bacteria such as Alcaligenes eutrophus, A. latus, Azotobacter vinlandii, and Zoogloea ramigera. Long pendant group PHAs are produced for example by many different Pseudomonas bacteria. Their biosynthesis involves the xcex2-oxidation of fatty acids and fatty acid synthesis as routes to the hydroxyacyl-coenzyme A monomeric units. The latter then are converted by PHA synthases which have substrate specificities favoring the larger C6-C14 monomeric units (Peoples and Sinskey, 1990).
In the case of the PHB-co-HX copolymers which usually are produced from cells grown on fatty acids, a combination of these routes can be responsible for the formation of the different monomeric units. Indeed, analysis of the DNA locus encoding the PHA synthase gene in Aeromonas caviae, which produces the copolymer PHB-co-3-hydroxyhexanoate, was used to identify a gene encoding a D-specific enoyl-CoA hydratase responsible for the production of the D-xcex2-hydroxybutyryl-CoA and D-xcex2-hydroxyhexanoyl-CoA units (Fukui and Doi, J. Bacteriol. 179:4821-30 (1997); Fukui et. al., J. Bacteriol. 180:667-73 (1998)). Other sources of such hydratase genes and enzymes include Alcaligenes, Pseudomonas, and Rhodospirillum.
The enzymes PHA synthase, acetoacetyl-CoA reductase, and xcex2-ketothiolase, which produce the short pendant group PHAs in A. eutrophus, are coded by an operon comprising the phbC-phbA-phbB genes; Peoples et al., 1987; Peoples and Sinskey, 1989). In the Pseudomonas organisms, the PHA synthases responsible for production of the long pendant group PHAs have been found to be encoded on the pha locus, specifically by the phaA and phaC genes (U.S. Pat. Nos. 5,245,023 and 5,250,430; Huisman et. al., J. Biol. Chem. 266:2191-98 (1991)). Since these earlier studies, a range of PHA biosynthetic genes have been isolated and characterized or identified from genome sequencing projects. Known PHA biosynthetic genes include: Aeronomas caviae (Fukui and Doi, 1997, J. Bacteriol. 179:4821-30); Alcaligenes eutrophus (U.S. Pat. Nos. 5,245,023; 5,250,430; 5,512,669; and 5,661,026; Peoples and Sinskey, J. Biol. Chem. 264:15298-03 (1989)); Acinetobacter (Schembri et. al., FEMS Microbiol. Lett. 118:145-52 (1994)); Chromatium vinosum (Liebergesell and Steinbuchel, Eur. J. Biochem. 209:135-50 (1992)); Methylobacterium extorquens (Valentin and Steinbuchel, Appl. Microbiol. Biotechnol. 39:309-17 (1993)); Nocardia corallina (GENBANK Accession No. AF019964; Hall et. al., 1998, Can. J. Microbiol. 44:687-69); Paracoccus denitrificans (Ueda et al., J. Bacteriol. 17:774-79 (1996); Yabutani et. al., FEMS Microbiol. Lett. 133:85-90 (1995)); Pseudomonas acidophila (Umeda et. al., 1998, Applied Biochemistry and Biotechnology, 70-72:341-52); Pseudomonas sp. 61-3 (Matsusaki et al., 1998, J. Bacteriol. 180:6459-67); Nocardia corallina; Pseudoinonas aeruginosa (Timm and Steinbuchel, Eur. J. Biochem. 209:15-30 (1992)); P. oleovorans (U.S. Pat. Nos. 5,245,023 and 5,250,430; Huisman et. al., J. Biol. Chem. 266(4):2191-98 (1991); Rhizobium etli (Cevallos et. al., J. Bacteriol. 178:1646-54 (1996)); R. meliloti (Tombolini et. al., Microbiology 141:2553-59 (1995)); Rhodococcus ruber (Pieper-Furst and Steinbuchel, FEMS Microbiol. Lett. 75:73-79 (1992)); Rhodospirillum rubrum (Hustede et. al., FEMS Microbiol. Lett 93:285-90 (1992)); Rhodobacter sphaeroides (Hustede et. al., FEMS Microbiol. Rev. 9:217-30 (1992); Biotechnol. Lett. 15:709-14 (1993); Synechocystis sp. (DNA Res. 3:109-36 (1996)); Thiocapsiae violacea (Appl. Microbiol. Biotechnol. 3:493-501 (1993)) and Zoogloea ramigera (Peoples et. al., J. Biol. Chem. 262:97-102 (1987); Peoples and Sinskey, Molecular Microbiology 3:349-57 (1989)). The availability of these genes or their published DNA sequences should provide a range of options for producing PHAs.
PHA synthases suitable for producing PHB-co-HH copolymers comprising from 1-99% HH monomers are encoded by the Rhodococcus ruber, Rhodospirillum rubrum, Thiocapsiae violacea, and Aeromonas caviae PHA synthase genes. PHA synthases useful for incorporating 3-hydroxyacids of 6-12 carbon atoms in addition to R-3-hydroxybutyrate i.e. for producing biological polymers equivalent to the chemically synthesized copolymers described in PCT WO 95/20614, PCT WO 95/20615, and PCT WO 95/20621 have been identified in a number of Pseudomonas and other bacteria (Steinbuichel and Wiese, Appl. Microbiol. Biotechnol. 37:691-97 (1992); Valentin et al., Appl. Microbiol. Biotechnol. 6:507-14 (1992); Valentin et al., Appl. Microbiol. Biotechnol. 40:710-16 (1994); Lee et al., Appl. Microbiol. Biotechnol. 42:901-09 (1995); Kato et al., Appl. Microbiol. Biotechnol. 45:363-70 (1996); Abe et al., Int. J. Biol. Macromol. 16:115-19 (1994); Valentin et al., Appl. Microbiol. Biotechnol. 46:261-67 (1996)) and can readily be isolated as described in U.S. Pat. Nos. 5,245,023 and 5,250,430. The PHA synthase from P. oleovorans (U.S. Pat. Nos. 5,245,023 and 5,250,430; Huisman et. al., J. Biol. Chem. 266(4): 2191-98 (1991)) is suitable for producing the long pendant group PHAs. Plant genes encoding xcex2-ketothiolase also have been identified (Vollack and Bach, Plant Physiol. 111:1097-107 (1996)).
Despite this ability to modify monomer composition by selection of the syntheses and substrates, it is desirable to modify other features of polymer biosynthesis, such as fatty acid metabolism.
It is therefore an object of the present invention to provide a method and DNA constructs to introduce fatty acid oxidation enzyme systems for manipulating the cellular metabolism of plants.
It is another object of the present invention to provide methods for enhancing the production of PHAs in plants, preferably in the oilseeds thereof.
Methods and systems to modify fatty acid biosynthesis and oxidation in plants to make new polymers are described. Two enzymes are essential: a hydratase such as D-specific enoyl-CoA hydratase, for example, the hydratase obtained from Aeromonas caviae, and a xcex2-oxidation enzyme system. Some plants have a xcex2-oxidation enzyme system which is sufficient to modify polymer synthesis when the plants are engineered to express the hydratase. Tissue specific and constitutive promoters were used to regulate and direct polymer production. Fusion constructs enhance polymer production.
Examples demonstrate production of polymer by expression of these enzymes in transgenic plants. Examples also demonstrate that modifications in fatty acid biosynthesis can be used to alter plant phenotypes, decreasing or eliminating seed production and increasing green plant biomass, as well as producing PHAs. Use of the phaseolin promoter can be used to induce male sterility. Tissue specific promoters in fusion constructs were used to modify production within regions of the seeds.